Herbal Materials
Herbs in THF [ Radix Notoginseng and Rhizoma Coptidis] were provided by Zhixin Chinese Herbal Medicine Co., Ltd. (Guangzhou, China. S). Professor Jiao Guo, Guangdong Pharmaceutical University authenticated the plant material using the Pharmacopeia of the people’s republic of China identification key (ISBN 2020, volume I). The production batch numbers are 210401 and 210501. Plant names have been checked with http://www.theplantlist.org.
Preparation And Chemical Constituents Of Thf
THF was prepared as follows[18], powdered Radix Notoginseng (400 g) and Rhizoma Coptidis (400 g) were separately extracted triply with 70% ethanol at 80 ℃ under reflux, each time for 2h. The extract solution was concentrated in a rotary evaporator to remove ethanol, and then dissolved in water and purified using D101 macro-porous resin (Lanxiao, Xi’an). The resulting purified extract was dried in vacuum at 60℃. The quantitative profiling of THF was performed on an U3000 HPLC with a DAD detector (Dionex, USA). The chromatograph separation was carried out using a Kromasil C18 column (4.5×250 mm, 5µm in particle size) according to the Pharmacopoeia of People’s Republic of China (2020), and data were recorded and analyzed on Chromeleon Console workstation. Finally, the content of eight active components in THF, namely, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rd, ginsenoside Re, notoginsenoside R1, berberine, coptisine, and palmatine, were quantified.
Animals And Management Ethics Protocol
Animals and management ethics protocol
All the animal experiments were approved by the Animal Ethical Committee of Guangdong Pharmaceutical University (SPF2017310). Specific pathogen free (SPF) male C57BL/6J Narl mice 3–4 weeks of age were purchased from Guangdong Medical Laboratory Animal Center. All mice were housed in a temperature-controlled room at 24°C ± 2°C, with a humidity of 60% − 70%, and 12 h of light and darkness alternated, standard solid food and water were provided during the experiment.
Induction Of Hyperglycemia In Experimental Animals
Mice were divided into two groups, normal and diabetic groups. To induce diabetes, mice were fed a HFD (60% fat, 20% protein, 20% carbohydrate; Research Diets, D12492) for 4 weeks [22]. Then, the mice were fasted for 6h followed by the administration of intraperitoneal (ip) injection of 40 mg/kg STZ for 4 consecutive days based on the previous study by Gilbert ER et al. [23]. The STZ (Sigma, St. Louis, MO, USA) was dissolved in citrate buffer (0.05 M; pH 4.5), which was freshly prepared before use. Blood glucose level was checked using an Accu-Chek blood glucometer (Roche Diagnostics, Basel, Switzerland) every 72 h. Stable hyperglycemia was established if the fasting blood glucose (FBG) was ≥ 12 mmol/L in the tested animals after 1 week from the STZ injection. Normal mice received i.p. injection of citrate buffer.
Mice were allocated randomly after 1 week from diabetes induction into three groups (n = 8) whereas the normal mice were allocated randomly after 1 week from ip injection of citrate buffer (n = 8) and treated daily as follows for 6 weeks. Control group: normal mice received oral normal saline only. Model group: animals were fed a HFD and received STZ. High-lose THF (THF-H) group: HFD/STZ diabetic mice were treated with THF (120 mg/kg/day). Low-lose THF (THF-L) group: HFD/STZ diabetic mice were treated with THF (60 mg/kg/day) [18]. Metformin (MET) group: HFD/STZ diabetic mice were administered MET (250 mg/kg).
The body weight and food intake of the mice were assessed once per week. The fat mass was detected in the last week of the experiment using the Minispec LF90 Body Composition Analyzer (Bruker). For the Oral Glucose Tolerance Test (OGTT), HFD mice were given oral glucose (2 g/kg) after fasting for 6 hours to measure their glucose tolerance, and the blood glucose levels at different time points were detected using the Accu-Chek blood glucometer immediately after the initial injection of glucose, Blood sugar level. The AUC of the glucose level over time was calculated to evaluate the glucose tolerance ability of the mice[24].
After all the experiments, all mice were euthanized with 200 mg/kg pentobarbital sodium through intraperitoneal injection, blood samples were collected from the mouse orbital venous plexus and transferred into a collection tube, which was then centrifuged at 3500 rpm for 30 min at 4 ℃, serum samples were prepared and kept at -80 ℃[18]. The TC, TG, APN and insulin levels in serum using commercial kits according to the manufacturers' introductions. Adipose tissue was collected and stored at -80 ℃ until subsequent biochemical analyses and was fixed in 10% buffered formalin for histopathological examination[24]. Animal bodies were taken care of by the Animal Ethical Committee of Guangdong Pharmaceutical University.
Histopathological Screening
Adipose tissue specimens were fixed in 10% formalin for 24 h, dehydrated using gradual ethanol concentrations, and embedded in paraffin. Then, the paraffin- embedded specimens were sectioned into 5 µm thick sections and stained with hematoxylin and eosin (H༆E)[24]. The adipose tissue (1mm×1mm×1mm) was placed in a 2.5% glutaraldehyde fixative solution, fixed overnight at 4℃, dehydrated, and observed under an electron microscope. Slides were examined under a light microscope (magnification:×200; Eclipse E200-LED, Nikon, Tokyo, Japan).
Metabolic Rate Measurements
The mice’s metabolism was evaluated by the Comprehensive Lab Animal Monitoring System (CLAMS, Columbus Instruments) according to the manufacturer's instructions. The mice were placed in individual cages and acclimated to the monitoring system for 24h. The metabolic rate was evaluated by their carbon dioxide production (VCO2), oxygen consumption (VO2) and heat production over the next 24h, which were analyzed with the CLAX Research software package (CLAX Research; CLAMS, Columbus Instruments)[24].
Determination Of Mitochondrial Mmp, Atp And Ca Content In Vat And 3t3-l1 Adipocyte
Fresh visceral adipose tissue(100 mg)were washed with PBS, then cutted into pieces and added 10 times the amount of pre-cooled mitochondrial separation reagent A (Biyuntian Biotechnology Co., Ltd., Shanghai, China), homogenized at low temperature, centrifuged to obtain the precipitate. The separated mitochondria in VAT and 3T3-L1 adipocytes were added to the mitochondrial storage solution and to detect the contents of MMP with the JC-1 Mitochondrial Membrane Potential Detection Kit (Biyuntian Biotechnology Co.Ltd., Shanghai, China), ATP (Biyuntian Biotechnology Co., Ltd., Shanghai, China) and mCa2+ (Jiemei Gene Pharmaceutical Technology Co., Ltd., China Shanghai) according to the kit instructions.
Cell Culture And Induction Of Adipocyte Differentiation
3T3-L1 preadipocytes were cultured and differentiated into adipocytes by using a previously reported method [25]. Briefly, 3T3-L1 preadipocytes were cultured in DMEM containing 10% bovine calf serum at 37℃in a 5% CO2 incubator. To induce differentiation, 2-day post confluent preadipocytes were incubated for 2 days in a differentiation medium containing 10% FBS, 0.5mM IBMX, 1µM dexamethasone, and l µg/mL insulin. The medium was then changed to DMEM containing 10% FBS and 1 µg/mL insulin, and cells were cultured for another 2 days. Following this, cells were incubated in DMEM supplemented with 10% FBS for 2 more days.
Rna Extraction And Quantitative Real-time Pcr
Total RNA was extracted using Trizol reagent (Takara, Dalian, China). The RNA was transcribed into cDNA using a reverse transcription kit (Takara, Dalian, China) the according to manufacturer's instructions, and quantitative real-time PCR was performed using a Light Cycler 480 real-time PCR system (Roche, Switzerland). β- actin was used as an internal control to normalize expression values. The sequences of the PCR primers are listed in Supporting Table 1.
Protein Extraction And Western Blotting Analysis
Western blotting analysis was performed according to the protocol as previously reported [16]. Protein was extracted by RIPA lysis buffer (P1003B, Beyotime, China) and protein concentration was detected by the BCA kit (P0011, Beyotime, China). The primary antibodies were incubated at 4℃overnight, and the information of the brand and item number of the primary antibody are listed in Supporting Table 2. After incubating proper secondary antibodies, the protein bands were visualized by enhanced chemiluminescence (ECL) kit (Bio-Rad, CA, USA) and quantified by Image Pro Plus software.
Statistical Analysis
Data are presented as means ± standard error of mean (SEM). Data sets that involved more than two groups were assessed by one-way ANOVA followed by Newman-Keuls post hoc tests. p < 0.05 was considered statistically significant. Graphpad Prism 6.0 software (GraphPad, CA, USA) was used for statistical analysis and graphics.